At least half the proteins encoded by the human genome are N-glycosylated, an essential post-translational modification. Defects in glycosylation underlie more than 100 human genetic disorders. For example, I-cell disease is caused by the inability to construct mannose-6-phosphate epitopes on N-glycans of lysosomal hydrolases, resulting in their secretion from cells rather than localization to lysosomes. Congenital Disorders of Glycosylation (CDGs), a family of severe inherited diseases with neurological and other symptoms, frequently result from defects in protein N-glycosylation. Major gaps remain in our understanding of basic glycosylation pathways. For example, assembly of the oligosaccharide donor for N-glycosylation requires flipping of three different glycolipids (Man5GlcNAc2-PP- dolichol (M5-DLO), mannose phosphate dolichol (MPD) and glucose phosphate dolichol (GPD)) from the cytoplasmic to the luminal side of the ER. These glycolipids have long hydrocarbon tails and very polar head groups. They represent key intermediates in the transition of the N-glycan biosynthetic pathway from the cytoplasmic to the luminal side of the ER. How they are flipped across the ER is a long-standing mystery. While there is compelling evidence that specific ER membrane proteins (flippases) are required, and that they have exquisite specificity for the lipids that they transport, the molecular identity of he flippases is not known. Our goal in this R21 application is to identify the glycolipid flippases responsible for flipping M5-DLO and MPD. We developed methods that recapitulate M5-DLO and MPD flipping in synthetic lipid vesicles and this technology now provides the cornerstone of the strategy that we will deploy to achieve our goal. We will use an innovative quantitative proteomics approach to identify flippase candidates from amongst yeast ER membrane proteins. We will then use our reconstitution-based assays to screen the candidates and evaluate their activity. This will provide conclusive evidence of function that we will corroborate by in vivo tests using yeast genetics. With this strategy we expect to identify, for the first time the flippases themselves. As our approach bypasses the limitations of traditional genetic and biochemical approaches that have thus far failed to provide the molecular identity of these flippases, we believe that we have a unique opportunity to solve this decades-old problem. Our discovery will contribute to basic cell biology by revealing a new class of transport proteins, associated with an undoubtedly novel transport mechanism, and also point to new genetic loci that are associated with CDGs.